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DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 2819
Development 137, 2819-2828 (2010) doi:10.1242/dev.052944
© 2010. Published by The Company of Biologists Ltd
Arginine methylation of SmB is required for Drosophila germ
cell development
Joël Anne*
SUMMARY
Sm proteins constitute the common core of spliceosomal small nuclear ribonucleoproteins. Although Sm proteins are known to
be methylated at specific arginine residues within the C-terminal arginine-glycine dipeptide (RG) repeats, the biological relevance
of these modifications remains unknown. In this study, a tissue-specific function of arginine methylation of the SmB protein was
identified in Drosophila. Analysis of the distribution of SmB during oogenesis revealed that this protein accumulates at the
posterior pole of the oocyte, a cytoplasmic region containing the polar granules, which are necessary for the formation of
primordial germ cells. The pole plasm localisation of SmB requires the methylation of arginine residues in its RG repeats by the
Capsuléen-Valois methylosome complex. Functional studies showed that the methylation of these arginine residues is essential for
distinct processes of the germline life cycle, including germ cell formation, migration and differentiation. In particular, the
methylation of a subset of these arginine residues appears essential for the anchoring of the polar granules at the posterior
cortex of the oocyte, whereas the methylation of another subset controls germ cell migration during embryogenesis. These
results demonstrate a crucial role of arginine methylation in directing the subcellular localisation of SmB and that this
modification contributes specifically to the establishment and development of germ cells.
INTRODUCTION
The small uridyl-rich nuclear ribonucleoproteins (U snRNPs) are
the major constituents of the spliceosome, a multicomponent
complex that catalyzes the splicing of pre-mRNAs in the nucleus.
The majority of U snRNP particles consist of one U snRNA,
several snRNA-specific proteins, and a common core of seven Sm
proteins. The loading of the Sm proteins onto the snRNA is
mediated by the survival of motor neurons (SMN) complex
(Meister and Fischer 2002; Massenet et al., 2002). The 7methylguanosine cap of the snRNA is then hypermethylated to a
2,2,7-trimethylguanosine cap (Mattaj, 1986) and the snRNP is
imported into the nucleus (Massenet et al., 2002; Narayanan et al.,
2004; Ospina et al., 2005).
Sm proteins are primarily small proteins that assemble to form
characteristic homomeric or heteromeric rings that consist of six or
seven subunits with a characteristic central pore, the presence of
which is crucial for binding U-rich regions of single-stranded RNA
(Scofield and Lynch, 2008). Members of the family are
characterised by a conserved bipartite Sm domain or ‘Sm fold’,
which functions, at least in part, in binding to neighbouring Sm
proteins within such rings (Hermann et al., 1995; Séraphin, 1995;
Khusial et al., 2005). SmB, SmD1 and SmD3 contain in their Cterminal tails glycine-arginine-glycine (GRG) and arginine-glycine
(RG) motifs, the arginine residues of which are post-translationally
methylated, a modification that takes place in the cytoplasm
(Brahms et al., 2000).
Department of Developmental Genetics, Deutsches Krebsforschungszentrum, Im
Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
*Author for correspondence ([email protected])
Accepted 9 June 2010
In human cells, symmetrical dimethylarginine (sDMA)
modification of Sm proteins is carried out by two distinct type II
protein arginine methyltransferases (PRMTs): PRMT5 and PRMT7
(Gonsalvez et al., 2007). In agreement with inhibitor-based results
showing that the recruitment of Sm proteins to the SMN complex
is facilitated by sDMA modification (Brahms et al., 2001; Friesen
et al., 2001a), depletion of either enzyme interferes with the ability
of SMN to efficiently recognise the Sm proteins (Gonsalvez et al.,
2007). Thus, sDMA modification of Sm proteins serves an
important regulatory function in human cells.
Loss of capsuléen (csul; the fly PRMT5 orthologue) activity
results in hypomethylation of Sm proteins (Anne and Mechler,
2005; Gonsalvez et al., 2006; Anne et al., 2007). Despite a
reduction in sDMA modification of Sm proteins, homozygous csul
mutants are viable (Gonsalvez et al., 2006; Anne et al., 2007). In
csul animals, the major Sm-class snRNPs are expressed normally
(Gonsalvez et al., 2006), suggesting that csul is dispensable for
snRNP assembly in Drosophila. In addition, flies express an
orthologue of PRMT7, Dart7 (Art7, CG9882) (Boulanger et al.,
2004), which is also required for sDMA modification of Sm
proteins in vivo (Gonsalvez et al., 2008). Unlike csul, Dart7 is an
essential gene (Gonsalvez et al., 2008).
Attempts have been made to elucidate the function of the
arginine methylation of Sm proteins. In human cultured cells the
RG-rich tail of the SmD3 protein is required for snRNP assembly
and optimal nuclear import (Friesen et al., 2001b; Girard et al.,
2004; Narayanan et al., 2004). Moreover, a lack of arginine
methylation at the C-terminus of SmD3 exerts no effect on snRNP
assembly in the cytoplasm (Khusial et al., 2005). The requirement
for methylation in snRNP assembly has been recently tested in
Drosophila. The use of two distinct lines that synthesise modified
isoforms of SmD1, in which either the entire RG-rich tail is deleted
or the arginine residues are substituted by lysine, shows that in
Drosophila the RG-rich tail of SmD1 is required for cell viability,
whereas the symmetric dimethylation of C-terminal arginine
DEVELOPMENT
KEY WORDS: Drosophila, Arginine methylation, Sm proteins, Germ cells, Oogenesis
residues in SmD1 is required neither for viability nor for snRNP
assembly (Gonsalvez et al., 2008). These data suggest that the
sDMA modification of SmD1 is dispensable for snRNP particle
assembly. The role, if any, of the post-translational methylation of
Sm proteins therefore remains unresolved. The goal of the present
work was to determine the functional relevance of arginine
methylation of SmB during Drosophila development.
MATERIALS AND METHODS
Molecular biology
Plasmid constructs were generated by PCR amplification of the relevant
DNA segments (High Fidelity PRC Master, Roche), which were subcloned
into the p-{Pvas-GFP} (Sano et al., 2002) or p-{UASp} (Rorth, 1998)
vectors. The SmD3 cDNA plasmid was kindly provided by H. Schenkel
(DKFZ, Heidelberg, Germany). SmB and SmD1 cDNAs (BDGP EST
Project clones LD14049 and RE39488, respectively) were provided by the
Drosophila Genomics Resource Center (DGRC), Bloomington, IN, USA.
SmB constructs and mutagenesis
A combination of site-directed mutagenesis using the QuikChange Kit
(Stratagene) and PCR amplification using mutagenic oligonucleotides was
used to substitute arginine residues in the SmB coding sequence. Arginine
residues R1-7 (at positions 112, 130, 1145, 174, 181, 189 and 196 of the
SmB sequence) were modified to leucine (R1 and R3-6) or glycine (R2 and
R7).
Immunocytochemistry
For whole-mount immunostaining, the following antibodies were used: antiOsk from rabbit and rat (gift of A. Ephrussi, EMBL, Heidelberg, Germany),
anti-Vas from rat (gift of P. Lasko, McGill University, Montréal, Québec,
Canada), anti-Tud from rabbit [TUD56; gift of S. Kobayashi (Amikura et al.,
2001)], anti-symmetrical dimethylarginine (SYM10) from rabbit (Upstate),
and anti-human SmB/B⬘ from mouse (ANA125, Calbiochem; gift of R.
Bordonné, Université de Montpellier, France). DNA was counterstained with
DAPI (Molecular Probes). Immunoreactivity was detected with appropriate
Cy3-, Cy5- (Jackson ImmunoResearch) or Alexa Fluor 488- (Molecular
Probes) conjugated secondary antibodies (1:200). Actin was detected using
Alexa Fluor-phalloidin (Molecular Probes) at 1:300. Images were acquired
on a Nikon Eclipse C1si laser-scanning confocal microscope and processed
with Adobe Photoshop and ImageJ software.
RESULTS
Distribution of SmB during oogenesis
Previous studies (Gonsalvez et al., 2006; Anne et al., 2007) have
shown that the SmB protein is an in vivo substrate of the
Capsuléen-Valois (Csul-Vls) methylosome complex. To monitor
the distribution of SmB during oogenesis, the SmB coding
sequence was cloned in frame with that of GFP under the control
of the vasa (vas) germline-specific promoter (Sano et al., 2002) and
a series of transgenic lines was generated; all tested lines produced
similar amounts of GFP-SmB proteins in the ovaries (data not
shown). As the methylated form of SmB, but not the unmethylated
form, can interact in vitro with the germline-specific protein Tudor
(Tud) (Anne et al., 2007), the distribution of GFP-SmB was first
compared with that of Tud. In early stages of egg chamber
development, GFP-SmB predominantly accumulated in the nuclei
of nurse cells and oocyte (Fig. 1A-D) [see Spradling (Spradling,
1993) for a detailed description of egg chamber development].
From stage 9 onward, GFP-SmB was detected at the posterior pole
of the oocyte (Fig. 1D and see Fig. S1 in the supplementary
material). As described by Bardsley et al. (Bardsley et al., 1993),
in early egg chambers Tud was found to be enriched in the nuage
surrounding nurse cell nuclei and was present in the cytoplasm of
the oocyte. At stage 7-8, Tud was localised temporarily at the
anterior margin of the oocyte and from stage 9 became restricted
Development 137 (17)
to the pole plasm (Fig. 1A-D). Thus, before their common
occurrence in the pole plasm, the two proteins displayed distinct
distribution patterns. In particular, no GFP-SmB could be detected
in the nuage or at the anterior cortex of the oocyte (Fig. 1C and 1D
insets). In the oocyte nucleus of previtellogenic egg chambers,
GFP-SmB was detected in a spherical structure attached to the
karyosome (Fig. 1E), which may correspond to the Cajal body (or
endobody) (Liu et al., 2006).
Localisation of Sm proteins in the pole plasm
Next, whether other Sm molecules could be detected in the pole
plasm was investigated. As with SmB, transgenic lines producing
GFP proteins fused to SmD1 or SmD3 were established. Both Sm
proteins were selected because they contain sDMA residues in their
C-terminal tails. No accumulation of GFP-SmD1 could be detected
at the posterior pole of the oocyte of transgenic females (Fig. 2A),
but a significant amount of GFP-SmD3 was detected at this
location (Fig. 2B). To check whether the localisation of the GFPSmB fusion protein was similar to that of endogenous SmB,
ovaries were stained using a monoclonal antibody (ANA125)
directed against the human SmB/B⬘ (SNRPB – Human Genome
Nomenclature Database) proteins. Only one protein band of the
expected molecular mass was detected on a western blot using this
antibody, demonstrating its specificity for SmB (Fig. 2C).
Immunostained egg chambers showed an accumulation of
endogenous SmB at the posterior pole of the oocyte, where it colocalised with the polar granule organiser Oskar (Osk) (Fig. 2D).
The ANA125 antibody also detected the endogenous SmB protein
at the posterior pole of the early embryos (data not shown). These
data indicate that only a subset of the Sm proteins, such as SmB
and SmD3, localises in pole plasm.
Genetic requirement for SmB localisation in the
pole plasm
To examine the mechanism by which SmB becomes targeted to the
pole plasm, the distribution of GFP-SmB in distinct genetic
backgrounds was analysed. As osk plays a cardinal role in pole
plasm formation (Ephrussi and Lehmann, 1992), the localisation of
GFP-SmB was first investigated in osk oocytes. GFP-SmB was
found to be absent from the posterior pole in oocytes of osk stage
10 egg chambers (Fig. 3A). The osk gene encodes two protein
isoforms that are differentially distributed in the oocyte. The short
form, which is active in pole plasm assembly, is a core component
of the polar granules (Breitwieser et al., 1996), whereas the long
form, which is required for their cortical anchoring, is associated
with endocytic membranes along the posterior cortex (Vanzo et
al., 2007). The localisation of GFP-SmB in egg chambers
overexpressing one of the Osk isoforms was then investigated. The
short and long Osk isoforms are carried by the p-{UAS-osk-M1RK10} and p-{UAS-osk-M139L-K10} transgenes, in which the 3⬘
UTR of the osk mRNA has been replaced by that of K10 and the
initiation codon of the long Osk isoform substituted by a codon
encoding an arginine or a leucine residue, respectively (Markussen
et al., 1995). These transgenes, in combination with a p-{nos-Gal4}
germline driver, directed the synthesis of high levels of either Osk
isoform in both nurse cells and oocyte (Vanzo and Ephrussi, 2002).
Examination of p-{GFP-Sm-B}; p-{UAS-osk-M1R-K10} /+; p-{nosGal4} /+ ovaries showed co-localisation of GFP-SmB with short
Osk at the anterior margin of the oocyte (Fig. 3B, arrowheads),
indicating that the short form of Osk could recruit GFP-SmB and
consequently that SmB is a component of the polar granules.
Analysis of p-{GFP-Sm-B}; p-{UAS-osk-M139L-K10} /+; p-{nos-
DEVELOPMENT
2820 RESEARCH ARTICLE
SmB methylation and germline development
RESEARCH ARTICLE 2821
Gal4} /+ ovaries indicated that, in contrast to long Osk, which can
be detected along the oocyte cortex, GFP-SmB is not observed at
this location (see Fig. S2 in the supplementary material),
suggesting that the long form of Osk is unable to recruit GFP-SmB.
GFP-SmB distribution was also examined in two vas alleles. The
vasO11 allele produces a Vas protein that is deficient in helicase
activity (Liang et al., 1994), whereas vasQ7 synthesises no
detectable full-length protein (Tomancak et al., 1998). In vasO11
stage 10 egg chambers, GFP-SmB was distributed normally at the
posterior pole of the oocyte (Fig. 3C), suggesting that the
accumulation of GFP-SmB in the pole plasm is independent of Vas
helicase activity. By contrast, no GFP-SmB staining could be
detected at the posterior pole of vasQ7 oocytes (Fig. 3D, upper
panels), but a visible signal was associated with numerous punctate
particles distributed throughout the cytoplasm in both nurse cells
and oocyte of vasQ7 egg chambers (Fig. 3D, lower panels). In
addition, the intensity of the GFP-SmB signal was markedly
reduced in the Cajal body of the vasQ7 oocyte nucleus (Fig. 3D,
lower-right panel) by comparison with that observed in vasO11.
However, in vasO11 oocytes, the nuclear GFP-SmB-containing
structure was frequently found to be dissociated from the
karyosome or split into two particles (Fig. 3C, lower panels). These
data indicate that Vas is needed for SmB localisation in the Cajal
body and that the helicase activity of Vas is not required for SmB
localisation in pole plasm.
In tud egg chambers, GFP-SmB localisation at the posterior pole
of the oocyte was abolished (Fig. 3E), indicating that Tud also
plays an essential role in the accumulation of SmB in the pole
plasm.
Csul/Vls-dependent localisation of Sm proteins in
the pole plasm
To check whether arginine methylation is required for the
localisation of SmB in the pole plasm, the distribution of GFPSmB in vls and csul mutant egg chambers was examined. Both
genes encode crucial components of the methylosome and are
required for the assembly of the pole plasm (Anne and Mechler,
2005; Gonsalvez et al., 2006; Anne et al., 2007).
First, it was determined whether the GFP-SmB protein is a
methylation target, similar to the endogenous SmB protein. For this
purpose, western blots of ovarian protein extracts from wild-type
and csul or vls mutant females carrying the p-{GFP-SmB}
transgene were examined using SYM10 antibodies, which
specifically recognise proteins containing multiple sDMA-glycine
repeats (Boisvert et al., 2002). As shown in Fig. 4A, these
antibodies predominantly reacted with a protein band of equivalent
molecular mass to the endogenous SmB protein. In addition, they
reacted with a band of ~50 kDa, which corresponds to the
transgenic GFP-SmB fusion protein in the extract derived from p{GFP-SmB} flies. In ovarian protein extracts of p-{GFP-SmB};
csulRM and p-{GFP-SmB}; vls3 flies, the intensity of these two
bands was strongly reduced, if not barely detectable. These results
indicate that the methylation of GFP-SmB recapitulates that of
endogenous SmB and confirm that the GFP-SmB protein is a
methylation target of the Csul-Vls methylosome.
The distribution of GFP-SmB in csul and vls mutant egg
chambers was then analysed. In previtellogenic egg chambers,
GFP-SmB was similarly distributed in wild-type and mutant nurse
cell nuclei as well as in the Cajal body of the oocyte nucleus (Fig.
DEVELOPMENT
Fig. 1. Distribution of GFP-SmB during Drosophila oogenesis. The egg chamber is made up of a germline cyst surrounded by a monolayer of
somatic follicle cells. Owing to incomplete cytokinesis, the cystocysts remain connected to each other through specialised cytoplasmic bridges. The
oocyte is specified from one of these germline cells, whereas the remaining 15 cells differentiate into nurse cells. The oocyte, which is the posteriormost cell in the cyst, increases in size dramatically as oogenesis proceeds. In all panels, egg chambers are oriented with the posterior to the right.
(A-D)Distribution of GFP-SmB and Tud in stage 4 (A), 6 (B), 8 (C) and 10 (D) egg chambers. GFP-SmB accumulates in nuclei of nurse cells and
oocyte throughout egg chamber development. In stage 10 egg chambers, GFP-SmB localises at the posterior pole of the oocyte. Note that GFPSmB only co-localises with Tud in pole plasm (arrowhead). The insets show a single nurse cell nucleus, taken at a different focal plane to those
shown in the whole stage 10 egg chamber, illustrating the absence of GFP-SmB accumulation in the nuage. (E)In previtellogenic egg chambers,
GFP-SmB is detected in a dot in the oocyte nucleus that corresponds to the Cajal body (green in the overlay). This structure is attached to the
karyosome, which is stained by the DNA-specific dye DAPI (red in the overlay).
Fig. 2. A significant amount of GFP-SmD3, but not GFP-SmD1,
accumulates in the pole plasm. (A,B)No GFP-SmD1 could be
detected at the posterior pole of the oocyte in stage 10 egg chambers
(A), whereas enrichment of GFP-SmD1 takes place in the pole plasm
(B). Enlargements of the oocyte posterior region are provided on the
right. (C)Anti-human SmB/B⬘ antibody (ANA125) recognises a single
protein band with the expected molecular mass of SmB in Drosophila
ovarian protein extracts. (D)Endogenous SmB (green in the overlay) colocalises with Osk (red in the overlay) in the pole plasm of stage 10 egg
chambers.
4B-D). However, GFP-SmB was detected at the oocyte posterior
pole in wild-type but not in mutant csul or vls stage 10 egg
chambers (Fig. 4B⬘-D⬘).
Whether SmD3 methylation by the Csul-Vls complex is also
crucial for its pole plasm localisation was then examined. GFPSmD3 protein was found to be absent from the posterior pole of
the csul or vls mutant oocyte (data not shown).
These data thus demonstrate that the activity of the methylosome
is required for the localisation of at least two Sm proteins, SmB and
SmD3, in the pole plasm.
Arginine methylation in RG repeats is essential
for SmB localisation
To extend the above results, the question of whether SmB
localisation in the pole plasm is dependent on methylation of the
arginine residues present in the RG repeats was addressed. The
arginines located at positions 112, 130, 145, 174, 181, 189 and
196 in the SmB protein are present in RG dipeptide repeats and
can be potentially methylated. These arginines are numbered 17 from the N-terminus to simplify mutant nomenclature. To
investigate their role, a series of p-{GFP-SmB} transgenes was
constructed, modified as follows: (1) deletion of the C-terminal
tail from residue 108 (SmB1-107); (2) substitution of the seven
arginines by leucine (L) or glycine (G) (SmBR1-7); (3)
substitution of the three proximal arginines by L or G (SmBR1-3);
and (4) substitution of the four distal arginines by L or G
(SmBR4-7). All the modified sequences were inserted downstream
of the vas promoter (Sano et al., 2002). Western blot analysis
revealed that these transgenic flies produce similar amounts of
GFP fusion proteins (Fig. 5A, upper panel). As shown in Fig. 5A
(lower panel), SYM10 antibodies identified the endogenous
SmB protein as well as the full-length unmodified GFP-SmB
protein (1-199), but not the C-terminally deleted GFP-SmB1-107
Development 137 (17)
Fig. 3. Genetic requirement for SmB localisation during
oogenesis. (A)Pole plasm accumulation of GFP-SmB requires osk
activity. GFP-SmB (green in the overlay) is detected at the posterior pole
of the oocyte in wild-type (WT, top), but not in osk84/Df(3R)pXT103
(osk, bottom), egg chambers. (B)GFP-SmB is recruited by the short
isoform of Osk. Overexpression of the short form of Osk during
oogenesis leads to an accumulation of Osk (red in the overlay) and GFPSmB (green in the overlay) at the anterior margin of the oocyte
(arrowheads) in stage 10 egg chambers. (C)In vasO11 stage 10 egg
chambers, GFP-SmB can be detected in the pole plasm (top) and in the
oocyte nucleus, either as a dot detached from the karyosome (bottom
left) or as two dots (bottom right). (D)In vasQ7 stage 10 egg chambers,
no GFP-SmB signal was observed at the posterior pole (top). However,
GFP-SmB staining formed multiple dots dispersed in the nuclei and
cytoplasm of nurse cells but not in the Cajal body (bottom). (E)Pole
plasm accumulation of GFP-SmB requires tud activity. GFP-SmB
remained undetected at the posterior pole of the tud1 oocyte. Higher
magnifications of the posterior cortex of the oocyte are provided on the
right (C-E).
or the fully substituted GFP-SmBR1-7 proteins. For the partially
substituted GFP-SmBR1-3 and GFP-SmBR4-7 proteins, the
intensity of the signal was significantly decreased.
The ability of the modified GFP-SmB proteins to accumulate in
the pole plasm was then examined (Fig. 5B-F). In contrast to GFPSmB (1-199), which robustly accumulated in the pole plasm (Fig.
5B), the two methylation-deficient proteins (GFP-SmB1-107 and
GFP-SmBR1-7) were unable to localise at the posterior pole of the
oocyte (Fig. 5C,D). The two partially methylated proteins (GFPSmBR1-3 and GFP-SmBR4-7) displayed a moderate accumulation in
the pole plasm (Fig. 5E,F). To further examine the role of arginine
methylation in targeting SmB to the pole plasm another SmB
mutant protein, in which the arginines of the tail were changed to
lysine (GFP-SmBR1-7K), was expressed during oogenesis. The
conservative arginine-to-lysine substitution retains the positive
charge of the residue but removes its ability to be targeted by
arginine methyltransferases. In agreement with the above results,
DEVELOPMENT
2822 RESEARCH ARTICLE
Fig. 4. Pole plasm accumulation of SmB requires methylosome
function. (A)The level of GFP-SmB methylation is similar to that of
endogenous SmB. Western blot of ovarian protein extracts from wildtype and transgenic females expressing GFP-SmB were probed with
SYM10 antibodies (which detect symmetrical dimethylarginines). The
GFP-SmB transgene was introduced into wild-type, csulRM and vls3
backgrounds. The intensity of the GFP-SmB and endogenous SmB
SYM10-immunoreactive protein bands was strongly reduced in csulRM
and vls3 protein extracts. The intensity of the other SYM10immunoreactive bands serves as an internal loading control.
(B-D⬘) Distribution of GFP-SmB in wild-type (B,B⬘), csulRM (C,C⬘) and vls3
(D,D⬘) egg chambers. In stage 7 egg chambers (B-D), the GFP signal
was detected with a similar intensity in the nuclei of wild-type and
mutant germ cells (the oocyte nucleus is indicated by an arrow in B),
whereas in mutant stage 10 oocytes (C⬘,D⬘) no GFP-SmB signal could
be detected at the posterior pole.
lysine substitution was found to abrogate the posterior pole
localisation of SmB (Fig. 5H). These data demonstrate that the
methylation of the arginine residues located in the RG repeats is
essential for the localisation of SmB in the pole plasm.
Arginine methylation of SmB in RG repeats is
zygotically required for gonad formation
Next, the function of SmB methylation during fly development was
investigated. For this purpose, SmB, SmBR1-7 and SmBR1-7K cDNAs
were cloned into a p-{UASp} transformation vector (Rorth, 1998)
and transgenic strains were generated. The transgenes were
then introduced into a mutant SmBBG02775/CyO heterozygous
background and the flies were crossed with SmBl(2)SH0509/CyO
flies carrying a ubiquitous tubulin-Gal4 driver. The
SmBBG02775/SmBl(2)SH0509 combination (hereafter designated SmB
for brevity) could be used to test whether expression of the SmB
constructs restores viability. p-{UAS-SmB}, p-{UAS-SmBR1-7} and
p-{UAS-SmBR1-7K} transgenes were found to be able to restore
normal development of SmB animals, but the rescue efficiency was
lower with the p-{UAS-SmBR1-7} and p-{UAS-SmBR1-7K} transgenes
(~50% of the p-{UAS-SmB} transgene efficiency; Table 1). The
transheterozygous combination was used because each SmBmutated chromosome 2 contained additional second-site lethal
mutations (data not shown). These results suggest that methylation
of the RG repeats of SmB is not crucial for fly viability.
All rescued SmB flies with p-{UAS-SmBR1-7} or p-{UASSmBR1-7K} (collectively p-{UAS-SmBR1-7 or R1-7K}) were, however,
found to be fully agametic (data not shown). To determine the
developmental phase of defective gamete production, SmB; p{UAS-SmBR1-7 or R1-7K} embryos were collected and stained for Vas,
a germ cell-specific marker (Hay et al., 1988). Identification of
SmB; p-{UAS-SmBR1-7 or R1-7K} embryos was performed using
RESEARCH ARTICLE 2823
Fig. 5. Pole plasm accumulation of SmB depends on the arginine
residues in its RG-rich tail. (A,G)Western blots of ovarian protein
extracts from transgenic females synthesising GFP-SmB fusion proteins,
probed with anti-GFP antibodies. Anti-ribosomal p40 antibodies (p40) were used as loading control. Similar blots were probed with
SYM10 antibodies. The white square in the lower panel of A indicates
the position of the GFP-SmB1-107 band, as revealed by incubation of the
same blot with anti-GFP antibodies (not shown). No SYM10 reactivity
could be detected for GFP-SmB1-107, GFP-SmBR1-7 or GFP-SmBR1-7K,
whereas GFP-SmBR1-3 and GFP-SmBR4-7 gave a reduced signal.
(B-F,H) Distribution of the GFP-SmB proteins in stage 10 egg chambers.
The size and structure of SmB and its derivatives are schematically
depicted, with two Sm domains and the RG dipeptides shown as
orange boxes and circles, respectively. Substituted arginine residues are
shown as white (leucine or glycine) or red (lysine) circles. In contrast to
GFP-SmB, which robustly localised at the posterior pole of the oocyte
(B), GFP-SmB1-107, GFP-SmBR1-7 and GFP-SmBR1-7K proteins were absent
from the pole plasm (C,D,H), and GFP-SmBR1-3 and GFP-SmBR4-7
proteins displayed a weak enrichment at this location (E,F). Higher
magnifications of the posterior cortex of the oocyte are provided on the
right.
SYM10 antibodies. Control experiment showed that, by
comparison with SYM10-positive wild-type and heterozygous
embryos, the methylation-deficient SmB; p-{UAS-SmBR1-7 or R1-7K}
embryos displayed only a weak signal (data not shown). In
wild-type embryos, the precursors of the germ cells cross the
primordial midgut epithelium and migrate bilaterally to reach the
gonadal mesoderm on the dorsal surface of the midgut.
Subsequently, germ cells associate, align and coalesce with the
gonadal mesoderm to form the gonads [see Campos-Ortega and
Hartenstein (Campos-Ortega and Hartenstein, 1985) for a detailed
description of embryonic developmental stages] (Moore et al.,
1998). Gonad formation was found to be abnormal in SmB;
p-{UAS-SmBR1-7 or R1-7K} embryos. Although germ cells could
DEVELOPMENT
SmB methylation and germline development
Development 137 (17)
Methylation of R4-7 in SmB is required for germ
cell development
The involvement of arginine residues R1-7 of SmB in gonad
formation prevents an analysis of their requirement for egg
chamber development. To circumvent this problem, it was reasoned
that decreasing the number of mutated arginines in SmB might
produce a residual activity enabling gonad formation and egg
chamber development. As the substitution of the arginines in the
last four RG repeats of SmB was shown to reduce the efficiency of
GFP-SmB targeting to the pole plasm, two transgenes, p-{UASSmBR4-7} and p-{UAS-SmBR4-7K}, were constructed and their in
vivo activity analysed. Expression of either transgene driven by
tubulin-Gal4 was able to rescue the zygotic lethality of SmB, albeit
with a lower efficiency than a wild-type p-{UAS-SmB} transgene
(Table 1). SmB; p-{UAS-SmBR4-7 or R4-7K} females developed
ovaries but they were reduced in size (Fig. 7A⬘; data not shown).
Ovary development in SmB; p-{UAS-SmB} females was similar to
that of wild type, with ovarioles forming chains of developing egg
chambers (Fig. 7B). By contrast, the ovaries of SmB; p-{UASSmBR4-7 or R4-7K} females contained predominantly previtellogenic
egg chambers (Fig. 7A⬘; data not shown) and rarely egg chambers
of later stages (see below). The majority of the egg chambers that
reached the end of previtellogenic development showed signs of
degeneration, including a condensed nuclear morphology (Fig. 7B⬘,
upper panel; data not shown).
To determine whether nuage formation could take place in early
SmB; p-{UAS-SmBR4-7 or R4-7K} egg chambers, a Tud staining was
performed. Tud was found to form a ring around the nurse cell
nuclei in these egg chambers (Fig. 7B⬘, lower panel; Fig. 8G),
suggesting that arginine methylation of SmB is not crucial for
nuage assembly (see also Anne et al., 2007).
These results indicate that a subset of arginine residues in the
SmB tail is necessary for the growth of the germ cells and
differentiation of the oocyte.
Fig. 6. SmB is zygotically required for gonad formation.
(A-B⬘) SmB; p-{UAS-SmB} (A,B) and SmB; p-{UAS-SmBR1-7} (A⬘,B⬘)
embryos were collected and stained using Vas antibodies, which
specifically label germ cells (red), and the DNA-specific dye DAPI
(green). (A)In stage 10 SmB; p-{UAS-SmB} embryos, the germ cells
migrated through the midgut epithelium, formed two bilateral clusters
within the mesoderm, and aligned with the somatic gonadal
precursors. (B)At stage 14, the two tissues coalesced to form the
embryonic gonads. In SmB; p-{UAS-SmBR1-7} embryos, the germ cells
were able to cross the epithelial wall of the midgut (A⬘), but were
unable to coalesce with the gonadal mesoderm in later stages (B⬘). In
SmB; p-{UAS-SmBR1-7}, the germ cells were scattered widely in the
posterior half of the embryo. The upper and lower panels in B⬘
represent sagittal sections of the same embryo at two different focal
planes.
Fig. 7. SmB is maternally required for egg chamber development.
(A-B⬘) Ovaries produced by SmB females rescued by p-{UAS-SmB} (A,B)
or p-{UAS-SmBR4-7} (A⬘,B⬘) driven by p-{tubulin-Gal4} were dissected
and stained for Tud (red) and for DNA with DAPI (green). By
comparison with SmB; p-{UAS-SmB} ovaries, which displayed normally
developing egg chambers (A), SmB; p-{UAS-SmBR4-7} ovaries were
underdeveloped and contained only a reduced number of
predominantly previtellogenic egg chambers (A⬘). A typical SmB; p{UAS-SmB} ovariole is made of developing egg chambers at different
stages (B), whereas the largest egg chambers in SmB; p-{UAS-SmBR4-7}
ovaries displayed signs of degeneration with pycnotic nurse cell nuclei
(B⬘, top). Nuage formation, as indicated by the perinuclear
accumulation of Tud protein, took place normally in SmB; p-{UASSmBR4-7} early egg chambers (B⬘, bottom).
Table 1. Rescue efficiency of wild-type and mutant SmB
transgenes
Transgene
WT
R1-7
R4-7
R1-7K
R1-3K
R4-7K
Rescue efficiency (%)
61 (n193)
32 (n238)
37 (n257)
28 (n174)
27 (n210)
30 (n171)
For the wild-type (WT) and mutant SmB transgenes, the percentage reflects the ratio
between the observed and expected number of homozygous flies.
migrate through the midgut epithelium (Fig. 6A) they were unable
to coalesce with the gonadal mesoderm. At stage 14 the Vaspositive germ cells were found to be dispersed in the posterior half
of the SmB; p-{UAS-SmBR1-7 or R1-7K} embryos. Few germ cells, at
most, were able to establish direct contact between them (Fig. 6B
and see Fig. S3A in the supplementary material). These results
show that SmB is zygotically required for the coalescence of germ
cells with the gonadal mesoderm and indicate that the methylation
of arginine residues in the tail of SmB is the key factor in this
process.
DEVELOPMENT
2824 RESEARCH ARTICLE
SmB methylation and germline development
RESEARCH ARTICLE 2825
Methylation of R4-7 in SmB is essential for polar
granule anchoring
As mentioned above, most of the SmB; p-{UAS-SmBR4-7} ovaries
contain only previtellogenic egg chambers. However, a small
fraction of the egg chambers was able to complete development in
the rescued SmB; p-{UAS-SmBR4-7} females, which laid few eggs.
These escapers allowed an analysis of the role of SmB methylation
in pole plasm formation in the oocytes as well as in pole cell
formation in the early embryos. By comparison with eggs laid by
SmB; p-{UAS-SmB} females that formed blastoderm embryos with
pole cells at their posterior pole (Fig. 8A), the embryos produced
by SmB; p-{UAS-SmBR4-7} females built a normal layer of
blastoderm cells but failed to form pole cells (Fig. 8A⬘).
Furthermore, the amount of Vas protein localised at the posterior
pole of early embryos derived from SmB; p-{UAS-SmBR4-7}
females was significantly reduced compared with embryos
produced by SmB; p-{UAS-SmB} females at similar stages (data not
shown). Examination of vitellogenic SmB; p-{UAS-SmB} egg
chambers revealed that, similarly to wild type, the Osk protein,
which is only synthesised in the pole plasm (Markussen et al.,
1995; Rongo et al., 1995), formed a dense and narrow crescent that
was tightly anchored at the posterior pole of the oocyte (Fig. 8B,C).
By contrast, Osk was found in a thicker structure that was loosely
attached to the posterior cortex and in particles that were dispersed
in the bulk ooplasm in SmB; p-{UAS-SmBR4-7 or R4-7K} oocytes at
similar stages of development (Fig. 8B⬘,C⬘,F).
A possible explanation of the failure to assemble Osk-containing
granules at the oocyte posterior pole is a defective cytoskeleton
network. To investigate this possibility, the distribution of the cortical
matrix of F-actin in vitellogenic oocytes was analysed. In SmB; p{UAS-SmB} oocytes, the F-actin was aligned as a continuous layer
underneath the oocyte cortex (Fig. 8C), whereas in SmB; p-{UASSmBR4-7 or R4-7K} oocytes the layer of F-actin appeared to be more
diffuse at the posterior pole (Fig. 8C⬘,F). Examination of Tud,
another pole plasm component, showed that its distribution bore the
same relationship to that of F-actin as did Osk (Fig. 8D⬘). To assess
whether Tud associates with Osk-containing particles, both proteins
were simultaneously detected in SmB; p-{UAS-SmBR4-7} oocytes.
Osk and Tud were found to co-localise in the released particles (Fig.
8E⬘). These data indicate that the methylation of arginine residues
R4-7 of SmB is necessary for the anchoring of the polar granules at
the posterior pole of the oocyte.
DEVELOPMENT
Fig. 8. Arginine methylation of SmB is maternally required for
the anchorage of the polar granules at the posterior pole of the
oocyte and for pole cell formation. Embryos or egg chambers of
SmB mutant females expressing p-{UAS-SmB} (A-E), p-{UAS-SmBR4-7}
(A’-E’) or p-{UAS-SmBR4-7K} (F,G) stained with the indicated antibodies
and dyes. (A,A⬘) Pole cells formed at the posterior pole of blastoderm
embryos laid by SmB; p-{UAS-SmB} females (A), whereas no pole cell
could be detected in embryos laid by SmB; p-{UAS-SmBR4-7} females
(A⬘). Germ cells were stained for Vas (red) and DNA with DAPI (green).
(B,B⬘) In a stage 9 SmB; p-{UAS-SmB} egg chamber, Osk was tightly
anchored at the posterior pole in a crescent-shaped pattern (B, red in
the overlay), but was loosely associated with the oocyte posterior cortex
in a stage 9 SmB; p-{UAS-SmBR4-7} egg chamber (B⬘). (C-F)Higher
magnifications of the posterior cortex of SmB; p-{UAS-SmB} (C-E), SmB;
p-{UAS-SmBR4-7} (C⬘-E⬘) and SmB; p-{UAS-SmBR4-7K} (F) stage 10
oocytes. Egg chambers were stained for Osk (red in the overlay of
C,C⬘,F and green in the overlay of E,E⬘), Tud (D,D⬘,E,E⬘; red in the
overlay), F-actin (C,C⬘,D,D⬘; green in the overlay) and DNA (blue in the
overlay). In SmB; p-{UAS-SmB} oocytes, Osk and Tud particles formed a
narrow crescent under the continuous cortical actin layer, whereas in
SmB; p-{UAS-SmBR4-7} and SmB; p-{UAS-SmBR4-7K} oocytes the Osk and
Tud particles were diffusely distributed in the cortical region and the
actin organisation was severely disrupted at the posterior pole. Osk and
Tud co-localised in the cortical polar granules in SmB; p-{UAS-SmB} (E)
and in the loosely associated particles (arrowhead in E⬘ overlay) in SmB;
p-{UAS-SmBR4-7} oocytes (E⬘). Note the correlation between the
disorganised actin network and failure to maintain Osk particles at the
cortex: the region indicated by the brackets in F shows that when the
actin organisation seems unaffected, cortical localisation of Osk also
appears normal, whereas both actin network and cortical accumulation
of Osk are disrupted in the region outside the brackets. (G)In a stage 9
SmB; p-{UAS-SmBR4-7K} egg chamber, Tud was loosely associated with
the oocyte posterior cortex but the nuage accumulation of Tud
appeared unaffected.
Methylation of R1-3 in SmB is zygotically required
for gonad formation
The above results also raise the question of whether the number or
the location of methylated arginine residues in the SmB tail is
functionally crucial. To address this point, a p-{UAS-SmBR1-3K}
transgene was constructed and its in vivo activity analysed.
Expression of the transgene driven by tubulin-Gal4 was able to
rescue the zygotic lethality of SmB and with a similar efficiency to
that of p-{UAS- SmBR1-7K} (Table 1). However, SmB; p-{UASSmBR1-3K} animals were found to produce no gametes (data not
shown). The defect in gonad formation arose at the same
developmental stages as observed with the p-{UAS- SmBR1-7K}
transgene (see Fig. S3B in the supplementary material). The
position, rather than the number, of methylated arginine residues
thus appears to be crucial for SmB function during gametogenesis.
As summarised in Fig. 9, the specific role of two subsets of
arginine residues in the SmB tail can be defined: the R1-3 subset
is required during embryogenesis for gonad formation, whereas the
R4-7 subset is involved in pole cell formation by controlling polar
granule maintenance at the posterior pole of the oocyte.
DISCUSSION
Arginine methylation-dependent localisation of
Sm proteins in pole plasm
Histochemical analysis indicates that the Sm proteins are
predominantly present in the nucleus, but studies using cell
fractionation techniques reveal that Sm proteins can be isolated as
Sm rings in the cytoplasm. Here, it was determined that two
Drosophila Sm proteins can specifically accumulate in the pole
plasm during the final stage of oocyte differentiation until the
formation of pole cells in blastoderm stage embryos. Inclusion of
Sm proteins in cytoplasmic structures is a rather novel finding,
although they have been detected outside of the nucleus in the germ
cells of various species, including Caenorhabditis elegans (Barbee
et al., 2002), Xenopus laevis (Bilinski et al., 2004), rat (Moussa et
al., 1994; Toyooka et al., 2000) and mouse (Chuma et al., 2003).
The present data show that access of SmB and SmD3 to the pole
plasm depends on sDMA methylation. Although SmD1 contains
arginine residues that are the targets of sDMA methylation, an
accumulation of SmD1 in the pole plasm was not observed. This
indicates that sDMA-modified Sm proteins, detected as GFP fusion
proteins, are differentially targeted to the pole plasm. As sDMAmodified Sm proteins can interact with the Tud domains of SMN
(Côté and Richard, 2005) and Tud has been previously shown to
bind methylated SmB protein (Anne et al., 2007), it is possible to
envisage that Tud confers specificity to the transport of SmB to the
pole plasm. However, this hypothesis is nullified by the finding that
SmB and Tud are conjointly detected only when both proteins have
reached their destination at the posterior pole of the oocyte. Recent
data indicate that the transport machinery responsible for the pole
plasm localisation of SmB and SmD3 might correspond to a
cytoplasmic osk mRNA RNP (Gonzalvez et al., 2010).
SmB and SmD3 can form a stable association (Raker et al.,
1996) but the unmethylated form of GFP-SmB was not detected in
the pole plasm even in the presence of both endogenous SmB and
SmD3. This suggests that the SmB and SmD3 proteins are
independently transported to the pole plasm or that they are
transported as heteromeric complexes in which the methylation of
both Sm proteins is tightly coupled. In favour of a coupling
methylation of an SmB-SmD3 complex is the finding that
hypomethylation of SmD3 is accompanied by a decrease in SmB
methylation (Gonzalvez et al., 2010).
Development 137 (17)
Fig. 9. Role of arginine methylation in germline establishment
and development. The present study identifies two functional groups
of arginine residues in the tail of SmB: the first group includes the three
proximal arginine residues and is required for gonad formation during
embryogenesis, whereas the second group comprises the last four
arginine residues and is involved in at least two processes: development
of the egg chambers within the ovaries and polar granule anchoring in
the oocyte. As the phenotype associated with the loss of these arginine
residues in SmB is distinct from that produced by capsuléen or valois
mutations, it is likely that other arginine methyltransferases (such as
Dart7) actively participate in SmB methylation during oogenesis. The
existence of a methylated factor that is able to bind to Tud and is
necessary for its localisation in nuage and pole plasm is inferred from
the presence of Tud in the nuage of SmB ovaries expressing a
hypomethylated form of SmB and from our previous work (Anne et al.,
2007).
Tud is also required for SmB localisation in the pole plasm. In a
previous report, we determined that the methyltransferase activity
of Csul is necessary for the localisation of Tud at the posterior pole
of the oocyte (Anne et al., 2007). If one considers a possible
association between Tud and methylated SmB in the polar
granules, this might explain why unmethylated SmB proteins do
not strongly accumulate in pole plasm.
Zygotic function of SmB in germ cell migration
SmB is first detected in the pole plasm during the early stages of
vitellogenesis and persists at this location until pole cell formation.
The effect of an absence of SmB methylation could only be
uncovered when the maternal contribution of methylated SmB is
depleted. The first zygotic abnormality resulting from the arginine
substitution in the tail of SmB, and therefore from a defect in SmB
methylation, is exposed when the germ cells initiate their migration
towards the gonadal mesoderm during late embryogenesis. The
function of SmB during these processes remains elusive, but a
parallel can be drawn with the occurrence of Sm proteins in the
spreading initiation centres (SICs) during the initiation of cell
spreading (de Hoog et al., 2004). SICs contain numerous
ribonucleoprotein complexes and exist temporarily during the very
early stages of cell spreading as precursors of focal adhesions. Cellmatrix attachments play an essential role in many vital cellular
processes, including motility, differentiation and survival.
Drosophila germ cells leave the midgut and migrate towards the
lateral mesoderm, displaying a highly polarised morphology, with
a broad lagging edge and an unusually long and relatively stable
rear protrusion (Sano et al., 2005). Whether Sm proteins are present
in this cell protrusion remains to be determined.
DEVELOPMENT
2826 RESEARCH ARTICLE
What is the role of SmB in polar granule
anchorage?
After the transport of the osk mRNA to the posterior pole of the
oocyte, this transcript is translated from in-phase alternative
initiation codons into two isoforms: a long form and a short form.
Owing to its unique ability to promote pole cell formation, only the
short Osk isoform can direct the assembly of polar granules
(Markussen et al., 1995; Breitwieser et al., 1996). Once polar
granule components are recruited by the short form, they must be
retained in place to ensure proper assembly of the pole plasm and
their inheritance by the pole cells. Polar granule maintenance
requires the long Osk form, which is necessary for the efficient
anchoring of the short Osk form at the oocyte cortex (Markussen
et al., 1995; Rongo et al., 1995; Vanzo and Ephrussi, 2002).
Importantly, each Osk isoform exhibits a distinct localisation at the
posterior pole: the short Osk is mainly present in the polar granules
(Breitwieser et al., 1996), whereas the long Osk form is attached to
endocytic membranes (Vanzo et al., 2007). The anchoring of the
other pole plasm components to the cortex requires an Oskdependent induction of endocytic activity (Tanaka and Nakamura,
2008). Western blot analysis revealed that both isoforms of Osk are
synthesised in SmB; p-{UAS-SmBR4-7} ovaries (data not shown),
suggesting that the defective anchoring phenotype cannot be
attributed to a failure to produce the long Osk form.
Our results suggest that the absence of SmB in pole plasm
modifies the organisation of the F-actin cytoskeleton and affects its
ability to anchor polar granules at the posterior cortex. Although
disruption of the microfilaments by treatment with the actindepolymerising drugs cytochalasin D or latrunculin A exerts only
a mild effect on the posterior anchoring of Osk (Babu et al., 2004),
several lines of evidence suggest that the F-actin cytoskeleton
anchors polar granules to the subcortical posterior region of the
oocyte. In particular, the actin-binding proteins Moesin and Bifocal
are required for this process (Jankovics et al., 2002; Polesello et al.,
2002; Babu et al., 2004). The cortical localisation of Bifocal in the
oocyte depends on an intact F-actin cytoskeleton and Bifocal binds
directly to F-actin filaments in vitro (Babu et al., 2004), suggesting
that Bifocal acts to stabilise F-actin filaments. The endocytic
pathway may also function downstream of long Osk to anchor the
pole plasm components at the cortex by regulating the dynamics of
F-actin (Tanaka and Nakamura, 2008). Projections of F-actin into
the ooplasm appear to emanate from the F-actin bundles overlying
the posterior cortex of the oocyte. These projections are oskdependent and become detectable from stage 10 onward, when
anchoring is required, and are of sufficient length to span the
distance between the plasma membrane and the underlying polar
granules (Vanzo et al., 2007). In the SmB; p-{UAS-SmBR4-7 or R4-7K}
egg chambers, there is a correlation between the loose anchoring
of the polar granules and the disorganisation of the F-actin
cytoskeleton. Although the possibility that SmB is also present at
the cortex cannot be excluded at this stage, a role of SmB in the
polar granules in establishing a link between these granules and the
cortical cytoskeleton network is plausible. The existence of a
positive-feedback loop maintenance mechanism, in which polar
granules, possibly in concert with long Osk, enhance their own
anchoring at the posterior pole, has indeed been proposed (Vanzo
et al., 2007).
Acknowledgements
This work was performed in the laboratory of Bernard Mechler, whom I thank
for constant support. I thank R. Bordonné, A. Ephrussi, S. Kobayashi, P. Lasko,
A. Nakimura and the Bloomington Stock Center for providing reagents; G.
Gonsalvez and G. Matera for sharing unpublished results; the two reviewers
RESEARCH ARTICLE 2827
whose comments helped to improve the quality of the manuscript; and the
Nikon Imaging Center (U. Engel and C. Ackermann) at the University of
Heidelberg. This work was funded by the Deutsches Krebsforschungszentrum
(DKFZ).
Competing interests statement
The author declares no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.052944/-/DC1
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